The daily organization of retinal function relies on passive responses to changes in ambient light intensity but also on a complex endogenous circadian clock system. The primary hallmark of clocks is that they continue to run in constant environmental conditions (e.g., total darkness) with periods of approximately 24 h (circadian clocks), and are synchronized to environmental rhythms through external cues, such as the light/dark cycle. By interacting with the dopamine and melatonin neuromodulator systems that are involved in light/dark adaptation, the retinal clock provides a mechanism to anticipate the change in light intensity that occurs at dawn and dusk, thus helping the retina to adapt in a time- and energy-efficient manner at the transition times. Accumulative indirect evidence indicates that impairment of the circadian regulation of retinal physiology may contribute to photoreceptor cell death in some degenerative retinal diseases. Therefore, establishing the basis of circadian rhythmicity in the retina is essential for our understanding of retinal physiology and pathophysiology. To this end, determining the nature and precise location of the circadian clock that drives daily rhythmicity in the retina is crucial. Recent developments in the field indicate that the clock machinery is a cell-based mechanism that relies on a set of genes and proteins (the clock components). Although the clock components are expressed in retinal tissue, the molecular mechanism and the exact location of the mammalian retinal clock are still elusive in part because clock gene expression is widespread among retinal layers. These observations suggest that more than one clock may be located in the mammalian retina. A circadian clock is likely located in the photoreceptor cells where it drives the rhythmic synthesis and release of melatonin. However, it is still unclear whether the rhythmic activity of the dopaminergic amacrine cells is under the control of a clock located within the dopaminergic cells or whether it is driven by the melatonin rhythm. Because melatonin and dopamine strongly interact with each other, it is also unclear whether light entrains retinal rhythms via its effects on dopamine or on melatonin. Our central hypothesis is that a fully functional circadian clock is located in the dopaminergic amacrine cells. The proposed experiments will be conducted on isolated mouse neural retinas maintained in vitro for several days in constant environmental conditions. Using an HPLC technique to measure dopamine levels and in situ hybridization and immunocytochemistry to analyze clock component expression, we will determine 1) whether a clock in the mouse retina controls dopamine levels and whether this rhythm is dependent on the melatonin rhythm and/or the presence of the photoreceptors; 2) which clock components display self-sustained expression in vitro and are required for the dopamine rhythm to occur; and 3) whether light entrainment primarily affects the dopamine and/or the melatonin rhythm and/or the expression of specific clock elements. Retinas from melatonin-deficient, melatonin-proficient and genetically-modified mice will be used in each of the specific aims. PUBLIC HEALTH RELEVANCE: Completion of this research project will provide a better understanding of the cellular and molecular basis of the circadian clock in the mammalian retina and thus increase our knowledge of how the circadian clock controls day/night differences in retinal function. Impairment of circadian rhythmicity in the retina has been linked to photoreceptor cell death and therefore this study will provide fundamental insights into the mechanisms that underlie retinal diseases such as retinitis pigmentosa.